Figure 4. Border zone cardiomyocytes undergo a metabolic switch from mitochondrial OXPHOS to glycolysis.

(a) Succinate dehydrogenase (SDH) enzyme staining on a seven dpi heart section with injury area separated by dashed line. Quantification of SDH activity in remote zone (RZ), border zone (BZ) and injury area (IA). Scale bar indicates 100 μm. Error bars indicate mean and standard deviation. (b) Transmission electron microscopy (TEM) images of mitochondria in cardiomyocytes from the remote zone and the border zone of a 7 dpi injured heart. Note the disorganized and irregular shaped mitochondria in the border zone cardiomyocyte. Scale bar 500 nm (200 nm in inserts). Graphs show quantification of mitochondrial perimeter-to-area as a measurement for roundness and quantification of mitochondrial cristae density. * p-value<0.05. (c) In situ hybridizations for glycolytic genes hk1, pkma and pdk2a on sections of injured zebrafish hearts at 7 dpi. Dashed line indicates injury site. Scale bars indicate 100 μm. (d) Time-lapse multi-photon confocal images of whole heart. The heart was isolated at 7 dpi and incubated with 2-NBDG, a fluorescent glucose analogue, at t = 0. Dotted line indicates injury area. Arrows point to regions of the border zone. Scale bar represents 100 μm. (e) Confocal image of injured zebrafish hearts at 7 dpi stained for the lactate transporter MCT4 (green) and Tropomyosin (red). Dashed line indicates injury site.

Figure 4—figure supplement 1. Energy metabolism genes are differentially expressed between cluster 7 and cluster 2 cells.

(a) Plot showing differentially expressed genes between cluster 7 versus cluster 2 cells. Differentially expressed genes (p-value<0.05) are highlighted in red (upregulated in cluster 7 cells) and blue (upregulated in cluster 2 cells). Complete gene list can be found in Figure 1—source data 3. 771 genes were differentially expressed (p<0.05; adjusted p-value after Benjamini-Hochberg correction), of which 752 were specifically upregulated in cluster seven cardiomyocytes, including the border zone genes, nppa, nppb and mustn1b. (b) GO-terms for upregulated genes in cluster 7 and cluster two respectively (p<0.01). (c) Gene set enrichment analysis for glycolysis genes on all genes with differential expression between cluster 7 versus cluster 2 cells (p<0.05). (d) Log2 fold change of differentially expressed genes (cluster 7 vs cluster 2) with a function in energy metabolism. High relative expression in cluster seven depicted in red, high expression in cluster two depicted in blue. In situ hybridization for the glycolysis gene hexokinase1 (a,b) and the embryonic cardiac gene myomesin1b (c,d) on sections of injured zebrafish hearts at three dpi (a,c) and seven dpi (b,d). While hk1 expression in border zone cardiomyocytes was visible at 3 dpi and seven dpi, myom1b expression was visible at seven dpi but undetectable at 3dpi. (a’,b’,c’ and d’) show zoom-in of boxed areas in corresponding panels. Dashed line indicates the injury site.